Small molecule probes are powerful tools for studying biological systems and can also serve as lead compounds for developing new drugs to treat human diseases. The objective of this project is to synthesize novel pilot-scale libraries of small molecules that will be submitted to the NIH Small Molecule Repository (SMR) for screening against a wide range of biological targets in the Molecular Libraries Screening Center Network (MLSCN). To date, pharmaceutical companies and, hence, commercial library suppliers, have largely focused on a relatively small number of "druggable" biological targets and, as a result, increasingly narrow regions of chemical structure space that correlate with these targets. However, sequencing of the human genome has opened the door to using small molecules to investigate a vast array of exciting new targets, both to gain fundamental insights into biological processes and to evaluate new therapeutic strategies. To address this much broader range of biological targets through screening in the MLSCN, a wider variety of chemical structures must also be represented in the SMR. To meet this need, we propose to synthesize pilot-scale libraries that are based on specific, privileged substructures from biologically active natural products. These libraries will access biologically-relevant regions of chemical structure space that are currently underrepresented due to the challenges associated with direct screening of natural products. We will use stereoselective syntheses to provide libraries with diverse three-dimensional structures. We have also designed these libraries with aqueous solubility, chemical stability, and cell permeability in mind. Every library member will be labeled with a versatile chemical handle to allow end users to attach reporter tags as needed for specific screening experiments. Thus, the specific aims of this project are: (1) synthesize pilot-scale libraries of stereodiverse spiroketals with sidechains that probe diverse three-dimensional vectors; (2) synthesize pilot-scale libraries of conformationally and structurally diverse polyketide substructures and use reiterative fragment couplings to generate larger, more complex polyketides. Broad-based screening in the MLSCN will provide a powerful new arsenal of small molecule probes to study fundamental biological processes, and will also facilitate future efforts in drug development, by delineating new potential therapeutic targets and novel classes of molecules that can be developed into new drugs.